Generation of cellular asymmetry is an unsolved problem fundamental to biology. This research seeks to develop a molecular mechanistic understanding of how yeast cells orient and assemble axes of polarity during vegetative division by budding. Yeast cells polarize, bud, and divide in two spatial patterns: the axial pattern of haploids in which buds form adjacent to the previous site of cell division, and the bipolar pattern of diploids in which buds form at either cell pole. The first focus is an investigation of the function of Bud10/Ax12, a type I transmembrane protein, which directs haploid cells to bud in axial orientations. Aims 1 and 2 investigate the following questions: How are the localizations of axial bud-site-selection proteins (Bud3, Bud4, Bud10) temporally controlled in the cell cycle? How is Bud10 regulated to signal in haploids but not in diploids? What are the signaling targets controlled by Bud10? The second focus of this proposal is an investigation of the mechanism by which the conserved GTPase Cdc42 controls cytoskeletal polarization. Experiments are designed to identify novel Cdc42 targets or downstream pathway components, and to analyze the mechanism of action of newly described Cdc42 targets, Gic1 and Gic2. The problem of cell polarization has implications for understanding human physiology and disease. Cell polarization underlies the development of axons in the nervous system, cell-cell communication and killing in the immune response, and cell migration throughout the body. Derangements of cellular organization are strongly associated with tumor metastasis and invasiveness. Many of the molecules that control polarization in yeast are conserved in humans, therefore what is learned should guide the understanding of similar processes in human cells.